Method for determining deletions in hbv pre-s2 region

ABSTRACT

A method of detecting pre-S 2  deletion mutant LHBS is disclosed herein. The method comprises incubating a biological sample with a first antibody to captured HBS proteins; detecting the LHBS and WT LHBS bound to the immobilized first antibody, respectively; and calculating the amount of the pre-S 2  deletion mutant LHBS protein by subtracting the amount of the WT LHBS protein from that of the LHBS protein. Advantageously, by the method described herein, the amount of the pre-S 2  deletion mutant LHBS, a potential high-risk marker for HCC incidence in chronic HBV carriers and recurrence in HCC patients after hepatectomy surgery, in a biological sample may be easily calculated without mutual influence between the WT and pre-S mutant LHBS while reducing the labor-intensive process for cloning each gene product before analysis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/333,346, filed on Jul. 16, 2014, in the United States Patent andTrademark Office, which claims the benefit of U.S. ProvisionalApplication No. 61/846,764, filed on Jul. 16, 2013, the disclosure ofwhich is incorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention generally relates to a method of detecting pre-S₂deletion mutant large hepatitis B virus surface protein.

2. Description of the Related Art

Chronic hepatitis B virus infection is a major cause of hepatocellularcarcinoma (HCC) worldwide and its most important cause in Asia.Hepatitis B virus (HBV)-related HCC often occurs at the age of 40 orolder, suggesting that HBV may persist in carriers for decades beforeHCC actually develops. It is important for long-term monitoringhigh-risk markers in chronic HBV carriers to identify the ones that needfrequent follow-up or uptake prophylactic therapies. In addition,development of high-risk recurrence markers in HCC patients receivinghepatectomy surgery is also important for clinicians to identify theones with relatively worse prognosis, who need to uptake aggressivetreatments. Though up to now the methods (e.g. ultrasound) and tumormarkers (e.g. alpha-fetal protein) to diagnose HCC have beenestablished, the high-risk markers for HCC incidence and recurrence havenot been fully identified, given that HCC tumorigenesis is a complexprocess regulated by various crosstalks between host and viral factors.

The pre-S₂ deletion mutant large hepatitis B virus surface protein(LHBS), isolated from the type 2 ground glass hepatocyte (GGH), ishighly associated with HCC (1). GGH is prevalent in livers with chronicHBV infection and HBV-induced HCC tumors. Type I GGH are usuallyscattered singly in the hepatic lobules with the expression of densehomogeneous or “inclusion-like” pattern of HBS. This type of GGH usuallyappears starting at the early carrier stage or in patients with activediseases, frequently co-expresses with a nuclear or cytoplasmic coreantigen (2,3). Previous studies found that type I GGH harbors the LHBSpartially deleted of the pre-S₁ region and accumulates in ER lumen (4).On the other hand, type II GGH expresses a unique pattern of HBS at thecell margin or periphery (For GGH morphologies, see FIG. 1). Mostinterestingly and importantly, this type of GGH consistently clusters innodules and usually appear at the advanced or low replicative stages ofvirus replication, and are frequently associated with cirrhosis or HCC,which suggests that type II GGH may represent clonally-proliferated,adenomatous, or preneoplatic lesions of HCC (2,3). By dissecting thecirrhotic nodules containing type II GGH, the HBV genomes were clonallyproliferated and integrated, supporting the concept of a clonal oradenomatous lesion of type II GGH. This type of GGH displays a uniquepattern of “marginal” type surface antigens and harbors the LHBSpartially deleted of the pre-S₂ region, designated pre-S₂ mutant LHBS(3). The pre-S₂ mutant LHBS is highly expressed in most of theHBV-induced cirrhotic nodules through the stages of the pre-neoplasticlesions, early HCC, and large HCC tumors, which implies the importantrole of pre-S₂ mutant LHBS in the tumor progression of HCC. The pre-S₂deletion region clusters at the N-terminus of the pre-S₂ region,predominantly in the range of 40 to 60 nucleotides long, although somecases contain deletions of as short as 10 nucleotides and still presenttype II GGH morphology (3). Some of the pre-S₂ mutant LHBS also harborsa point mutation at the pre-S₂ ATG start codon, causing no synthesis ofthe middle S antigen (FIG. 2). The region commonly deleted in the pre-S₂mutant LHBS contains a CD8 T-cell epitope, which proposes a hypothesisthat this type of mutant LHBS escapes from immune surveillance thenbecomes a selective clone in long-term persistence of HBV infection.

In the past 10 years or so, the inventors of the present inventionfocused on studying the pathological effects caused by the pre-S mutantLHBS, especially the pre-S₂ type, since this type of LHBS isparticularly highly associated with HCC. It was found that the both ofthe pre-S₁ and -S₂ mutant LHBS distribute in endoplasmic reticulum (ER)of hepatocytes and induce ER stress (4). However, the pre-S₂ mutant LHBSspecifically induces ER stress-induced growth proliferation mediatedthrough the NF κ B pathway (5). The pre-S₂ mutant LHBS also inducessignificantly higher level of ER stress-dependent oxidative stress andoxidative DNA damages than the wild-type or pre-S₁ mutant LHBS do, whichleads to genomic instability and consequently hepatocellularcarcinogenesis (6). In addition, the pre-S₂ mutant LHBS specificallyinduces cyclin A over-expression (7) and directly interacts with Junactivation domain-binding protein 1 (JAB1) to cause inactivation of thetumor suppressor retinoblastoma and cell cycle progression (8). Togetherthese findings have offered clear lines of evidence of that the pre-S₂mutant LHBS likely plays an important role in HBV-induced HCC.

The pre-S₂ mutant LHBS is relatively low at acute phase of HBV infectionbut greatly increased in the long-term period of HBV infection. In HCC,it is higher than 60%, which indicates that it is highly prevalent inlate phase of chronic HBV infection and significantly associated withadvanced liver diseases such as cirrhosis and HCC (9). To detect thepre-S deletion in LHBS, we previously developed a Pre-S Gene Chip, whichdetects the pre-S deletion based on DNA hybridization of the HBS genesin patients to the DNA probes on the chip. It was found that the WT andpre-S mutant LHBS often co-exist in one individual HBV carrier, whichmakes the detection of the pre-S mutant difficult. Moreover, it requireslabor-intensive process for cloning and isolating each individual geneproduct before the chip analysis.

Furthermore, the pre-S₂ deletion mutants have also been shown toincrease after anti-viral therapies using nucleoside analogs to chronicHBV carriers, suggesting that the carriers presenting the pre-S₂ mutantlarge HBV surface (LHBS) protein are the ones in high risk to HCC. SeeZhang et al. BMC Microbiology, 12: 307-316 (2012). Thus, there is a needto develop a rapid and accurate method for detecting deletions in theHBV Pre-S region, thereby assessing a HBV carrier's risk of developingcirrhosis/HCC.

REFERENCES

1. Gerber M A, Hadziyannis S, Vernace S, Vissoulis C. Incidence andnature of cytoplasmic hepatitis B antigen in hepatocytes. Lab Invest.1975; 32:251-256.

2. Fan Y F, Lu C C, Chang Y C, Chang T T, Lin P W, Lei H Y, Su I J.Identification of a pre-S2 mutant in hepatocytes expressing a novelmarginal pattern of surface antigen in advanced disease of chronichepatitis B virus infection. J. Gastroenterol. Hepatol. 2000;15:519-528.

3. Fan Y F, Lu C C, Chen W C, et al. Prevalence and significance ofhepatitis B virus (HBV) pre-S mutants in serum and liver at differentreplicative stages of chronic HBV infection. Hepatology 2001;33:277-286.

4. Wang H C, Wu H C, Chen C F, et al. Different types of ground glasshepatocytes in chronic hepatitis B virus infection contain specificpre-S mutants that may induce endoplasmic reticulum stress. Am. J.Pathol. 2003; 163:2441-2449.

5. Hung J H, Su I J, Lei H Y, et al. Endoplasmic reticulum stressstimulates the expression of cyclooxygenase-2 through activation ofNF-kappaB and pp38 mitogen-activated protein kinase. J Biol Chem. 2004;279:46384-46392.

6. Hsieh Y H, Su I J, Wang H C, et al. The pre-S mutant surface antigensin chronic hepatitis B virus infection induce oxidative stress and DNAdamage. Carcinogenesis 2004; 25:2023-2032.

7. Wang H C, Huang W, Lai M D, et al. Aberrant cyclin A expression andcentrosome overduplication induced by hepatitis B virus pre-S2 mutantsand its implication in hepatocarcinogenesis. Carcinogenesis 2012;33:466-472.

8. Hsieh Y H, Su I J, Wang H C, et al. Hepatitis B virus pre-S2 mutantsurface antigen induces degradation of cyclin-dependent kinase inhibitorp27Kip1 through c-Jun activation domain-binding protein 1. Mol CancerRes 2007; 5:1063-1072.

9. Shen F C, Su I J, Wu H C, et al. A Pre-S Gene Chip to detect thepre-S deletions in the hepatitis B virus large surface antigen as apredictive marker for hepatoma risk in the chronic HBV carriers. J.Biomed. Sci. 2009; 16:84-91.

10. Shafritz D A, Shouval D, Sherman H I, et al. Integration ofhepatitis B virus DNA into the genome of liver cells in chronic liverdisease and hepatocellular carcinoma. Studies in percutaneous liverbiopsies and post-mortem tissue specimens. N Engl J Med 1981;305:1067-1073.

11. Guerrieri F, Belloni L, Pediconi N, Levrero M. Molecular mechanismsof HBV-associated hepatocarcinogenesis. Semin Liver Dis 2013;33:147-156.

12. Hung L, Kumar V. Specific inhibition of gene expression andtransactivation functions of hepatitis B virus X protein and c-myc bysmall interfering RNAs. FEBS Lett 2004; 560:210-214.

13. Becker S A, Lee T H, Butel J S, et al. Hepatitis B virus X proteininterferes with cellular DNA repair. J Virol 1998;72:266-272.

14. Chen B F, Liu C J, Jow G M, et al. High prevalence and mapping ofpre-S deletion in hepatitis B virus carriers with progressive liverdiseases. Gastroenterology 2006; 130:1153-1168.

15. Chen C H, Hung C H, Lee C M, et al. Pre-S deletion and complexmutations of hepatitis B virus related to advanced liver disease inHBeAg-negative patients. Gastroenterology 2007; 133:1466-1474.

16. Tsai H W, Lin Y J, Lin P W, et al. A clustered ground-glasshepatocyte pattern represents a new prognostic marker for the recurrenceof hepatocellular carcinoma after surgery. Cancer 2011; 117:2951-2960.

17. Zhang D, Dong P, Zhang K, et al. Whole genome HBV deletion profilesand the accumulation of preS deletion mutant during antiviral treatment.BMC Microbiol. 2012; 12:307.

SUMMARY OF THE INVENTION

Therefore, it is a primary objective of the present invention to providea method of detecting pre-S₂ deletion mutant LHBS using an immunoassaykit to evaluate the pre-S₂ mutant LHBS as a potential high-risk markerfor HCC incidence in chronic HBV carriers and recurrence in HCC patientsafter hepatectomy surgery.

To achieve the foregoing objective, the present invention provides amethod of detecting a pre-S₂ deletion mutant large hepatitis B virussurface protein in a biological sample, comprising: incubating abiological sample with a first antibody immobilized on a substrate tobind a hepatitis B virus surface protein to the first antibody, whereinthe first antibody specifically binds to amino acid residues 1-226 ofhepatitis B virus surface protein; detecting a large hepatitis B virussurface protein and a wild type large hepatitis B virus surface proteinbound to the immobilized first antibody by incubating the boundhepatitis B virus surface protein with a second antibody specificallybinding to amino acid residues 1-119 of large hepatitis B virus surfaceprotein and a third antibody specifically binding to amino acid residues125-142 of large hepatitis B virus surface protein, respectively; andcalculating the amount of the pre-S₂ deletion mutant large hepatitis Bvirus surface protein by subtracting the amount of the wild type largehepatitis B virus surface protein from that of the large hepatitis Bvirus surface protein.

Preferably, the biological sample may be isolated from a human.

Preferably, the human may be a HBV carrier or a liver cancer patient.

Preferably, the HBV carrier may be HBeAg-negative, with HBV viral loadhigher than 10⁴ copies/ml of the biological sample, or the combinationthereof.

Preferably, the method may further comprise a step of assessing a riskof developing hepatocellular carcinoma or cirrhosis of the HBV carrier.

Preferably, the HBV carrier may have undergone an antiviral therapyusing nucleoside analogs or interferon drugs.

Preferably, the method may further comprise a step of assessing a riskof developing hepatocellular carcinoma or cirrhosis after the antiviraltherapy.

Preferably, the liver cancer patient may be a hepatocellular carcinomapatient undergone a hepatectomy surgery.

Preferably, the method may further comprise a step of assessing risk ofcancer recurrence of the hepatocellular carcinoma patient at 1 to 12month(s) after the surgery.

Preferably, the biological sample may be whole blood, plasma or serum.

Preferably, the method may further comprise a step of isolatinghepatitis B virus surface protein from the biological sample.

The method of detecting pre-S₂ deletion mutant LHBS using theimmunoassay kit according to embodiments of the present invention mayhave the following advantages:

(1) The immunoassay kit comprising the antibodies and the method ofdetecting pre-S₂ deletion mutant LHBS using the same of the presentinvention may be easy to calculate the amount of the pre-S₂ deletionmutant LHBS in a biological sample without mutual influence between thewild-type and pre-S mutant LHBS.

(2) The immunoassay kit comprising the antibodies and the method ofdetecting pre-S₂ deletion mutant LHBS using the same of the presentinvention may reduce the labor-intensive process for cloning andisolating each individual gene product before analysis comparing toPre-S Gene Chip analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The properties and effects of the present invention will now bedescribed in more details hereinafter with reference to the accompanyingdrawings that show various embodiments of the invention as follows.

FIG. 1 is a schematic view of GGH and the expression patterns ofhepatitis B virus surface antigen. Type I GGH are usually scatteredsingly (a, hematoxylin-eosin [HE] stain ×200) and express aninclusion-like pattern of the surface antigen (b, immunohistochemicalstain for the surface antigen). Type II GGH consistently cluster innodules (c) and express a marginal pattern of the surface antigen (d).

FIG. 2 is a schematic view of the wild-type, pre-S₁ and pre-S₂ mutantLHBS gene. The shaded boxes are the regions deleted in the pre-S1 andpre-S₂ mutant LHBS, respectively. The numbers labeled on the bottom ofthe gene are to indicate the regions of the pre-S₁, pre-S₂ and S regionsof the gene in nucleotide sequences of HBV genome. The arrow on top ofthe diagram indicates the start (nt. 1) of the HBV genome.

FIG. 3 is a schematic view of detecting LHBS and WT LHBS using theimmunoassay kit according to embodiments of the present invention.

FIG. 4 demonstrates sensitivities and specificities of the LHBSantibody, detected by ELISA, according to embodiments of the presentinvention. (A) Binding of the LHBS antibody to various concentrations ofthe WT and pre-S₂ deletion mutant LHBS; and (B) Binding of varioustitrations of the LHBS antibody to the WT and pre-S₂ deletion mutantLHBS in the amounts of 50 ng.

FIG. 5 demonstrates sensitivities and specificities of the WTLHBS-specific antibody, detected by ELISA, according to embodiments ofthe present invention. (A) Binding of the WT LHBS-specific antibody tovarious concentrations of the WT and pre-S₂ deletion mutant LHBS; and(B) Binding of various titrations of the WT LHBS-specific antibody tothe WT and pre-S₂ deletion mutant LHBS in the amounts of 50 ng.

FIG. 6 is a schematic view of using the WT LHBS-specific antibodyaccording to embodiments of the present invention to detect the LHBS andthe WT LHBS. : WT only. ▴: WT/pre-S₂ deletion mutant LHBS mixed invarious relative ratios. The LHBS amount is 50 ng.

FIG. 7 is a schematic view of detection of LHBS in HBsAg(+) and HBsAg(−)patient sera. (A) The LHBS levels (ng/μl). (B) The ROC curve of theimmunoassay kit according to embodiments of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The technical content of the present invention will become apparent bythe detailed description of the following embodiments and theillustration of related drawings as follows.

Definitions

The term “HBS” as used herein refers to surface protein of hepatitis Bvirus (HBV). The HBS comprises large HBS, middle HBS and small HBS,which are different splicing forms of the surface protein.

The term “LHBS” as used herein refers to large surface proteincomprising pre-S₁, pre-S₂ and S regions.

The term “WT LHBS” as used herein refers to wild-type large surfaceprotein comprising pre-S₁, pre-S₂ and S regions with a length of 401amino acids.

The term “pre-S₂ deletion mutant LHBS” as used herein refers to largesurface protein with a deletion around 20 amino acids in the pre-S₂region.

The term “detecting” is used in the broadest sense to include bothqualitative and quantitative measurements of a target molecule. In oneaspect, the detecting method as described herein is used to identify themere presence of HBS, LHBS or WT LHBS in a biological sample. In anotheraspect, the method is used to test whether HBS, LHBS or WT LHBS in asample is at a detectable level. In yet another aspect, the method canbe used to quantify the amount of HBS, LHBS or WT LHBS in a sample andfurther to compare the HBS, LHBS or WT LHBS levels from differentsamples

The term “biological sample” refers to a body sample from any animal,but preferably is from a mammal, more preferably from a human. Mostpreferably, such biological sample is from HBV carrier or a liver cancerpatient. Such samples include biological fluids such as serum, plasma,vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminalfluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid,saliva, sputum, tears, perspiration, mucus, and tissue culture medium,as well as tissue extracts such as homogenized tissue, and cellularextracts, in which HBS may be detected. The preferred biological sampleherein is serum, plasma or whole blood.

The term “first antibody” refers to an antibody capable of binding andcapturing a target molecule, HBS, in a sample such that under suitablecondition, the first antibody-HBS complex can be separated from the restof the sample. Typically, the first antibody is immobilized orimmobilizable. In a sandwich immunoassay, the first antibody ispreferably an antibody, antigen-binding fragment, a functional variantor a mixture thereof against a HBS.

The terms “second antibody” and “third antibody” refer to an antibodythat is capable of being detected either directly through labeling asignal generating unit, or indirectly through, e.g., another antibodythat is labeled with the signal generating unit. The preferred secondantibody and third antibody are LHBS-specific antibody and WTLHBS-specific antibody, respectively.

The term “signal generating unit” refers to a moiety or technique usedto detect the presence of the second antibody and the third antibody inthe immunoassay herein and includes detection agents that amplify theimmobilized label such as label captured onto a microtiter plate.Preferably, the signal generating unit is a radioactive marker, afluorescent marker, a phosphorescent marker, a chemiluminescent markeror a labeling enzyme. More preferably, the signal generating unit is thelabeling enzyme comprising horse radish peroxidase or alkalinephosphatase.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including agonist, antagonist, and neutralizingantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies, and antibody fragments so long as they exhibitthe desired binding specificity.

Material and Methods

Development of Monoclonal Antibodies that Specifically Recognize LHBSand WT LHBS

To generate monoclonal antibodies specifically recognizing LHBS,briefly, inventors purified the recombinant pre-S region protein (SEQ IDNO: 1) from E. coli and i.p. injected it into BALB/C mice to generatemonoclonal antibodies. The activated splenocytes were fused withmelanoma cells to generate hybridomas. The hybridoma clones which showedthe highest affinities to the pre-S₁ region protein among all were i.p.injected into mice for ascites production to amplify the antibodies. Theantibody clones which showed the highest affinity to LHBS among all werefurther selected for further investigation. Preferably, the LHBSantibody may be produced by the hybridoma cell line deposited on Sep. 1,2014 under accession number BCRC960490 at the Food Industry Research andDevelopment Institute, 331 Shih-Pin Road, Hsinchu 300, Taiwan, and alsoon Oct. 31, 2014 under accession number DSM ACC3253 at Leibniz-InstitutDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ),Inhoffenstr. 7B, 38124 Braunschweig, Germany.

To generate the monoclonal antibodies specifically recognizing the WTLHBS, the amino acid sequence (SEQ ID NO: 2) of the protein region thatis deleted in the pre-S₂ deletion mutant LHBS has been synthesized intoa peptide and injected to mice for generating the WT LHBS-specificantibody. The mouse whose serum showed the highest affinities to thewild-type LHBS was selected for further investigation. Preferably, theWT LHBS antibody may be produced by the hybridoma cell line deposited onJul. 16, 2014 under accession number BCRC960488 at the Food IndustryResearch and Development Institute, 331 Shih-Pin Road, Hsinchu 300,Taiwan, and also on Oct. 31, 2014 under accession number DSM ACC3251 atLeibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany.The detail embodiments are described below.

Antigen Preparation for Mouse Immunization

Preparation for E. coli Cell Lysates

Recombinant protein, pre-S₁, protein sequence consulted from NCBI(National Center for Biotechnology Information), and hepatitis B virusserotype, adw2, were adapted for cloning template reference. Pre-S₁region (a.a. 1-119, SEQ ID NO: 1) of LHBS was expressed by E. coliprotein expression system with Rosetta strain. The pre-S₁ recombinantplasmid in E. coli cells was adapted from lab stock that pre-S₁ segmentwas designed to be cloned into plasmid, pET21c, with 6x-His tag genes atthe 5′ end of pre-S₁ gene. By transforming this plasmid into Rosettacells, then colonies were picked and culture with LB medium, 37° C.,until the OD₆₀₀ reach 0.6-0.8 around 16 hours; and refresh the culturein new batch of LB medium (pH 8) and incubate in 37° C. when OD₆₀₀ read0.8˜1.0; 100 mM IPTG is added to 0.2 mM final concentration for 6 hoursinduction in 25° C. Induction sample was then harvested by 6000 rpm, 10minutes centrifugation, the supernatant was removed, and the pellet wascollected for sonication to acquire cell lysates. The collected cellpellet was resuspended in 1× binding buffer (20 mM phosphate solution,pH 7.4, 0.5 M NaCl, and 20 mM Imidazole), and by sonication in 130 watt,20 kHz, 75% amplified burst for 30 seconds every time and for 3-4 times.Followed by 10000 rpm, 30 minutes centrifugation in 4° C. to discard thecell debris, the supernatant of cell lysates was acquired for Ni²⁺-NTAchromatography purification.

Protein Purification by Ni²⁺-Chelating Chromatography

Pre-S₁ recombinant protein purification was processed with packing Ni²⁺resin first by adding 2 resin volumes of 100 mM Ni²+ (100 mM NiSO₄) withproper volume of chelating resin. The column was then washed with 5resin volumes of MiniQ water, and conditioned with 5 resin volumes ofbinding buffer. Then, the E. coli cell lysates were added to the Ni²⁺column and then washed with washing buffer 1 (60 mM Immidazole, 20 mMsodium phosphate, 0.5 M sodium chloride, pH 7.4), washing buffer 2 (80mM Immidazole, 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.4),with each 10 resin volumes. Finally, eluted the target protein with 3resin volume of elution buffer (500 mM Immidazole, 20 mM sodiumphosphate, 0.5 M sodium chloride, pH 7.4), and then check proteinpurification quality and efficiency by SDS-PAGE electrophoresis andimmunoblotting on collected sample from each step. The pre-S₁recombinant protein was further purified by DEDE-ion exchange column toreach the criteria for mouse immunization.

Pre-S₁ Region Monoclonal Antibody (LHBS-Specific Antibody) Production

The pre-S₁ region recombinant protein, acquired from E. coli system, 0.5mg/ml, total around 3 mg was sent for commercial antibody production.The mouse immunization and hybridoma production were processed byProtech, Inc., Taiwan. Mice antisera can be acquired after 2.5 monthsfor antigen specific antibody titering to select an ideal mouse forsacrifice and hybridoma fusion experiment. Later, culture medium fromhybridoma can be also acquired for antigen specific antibody titeringseveral times to select the perfect clones for antigen specificmonoclonal antibody production. The hybridoma cells were then acquiredand isotyped to ensure these cells are monoclonal or yet and limitdilution required to get cells with monoclonal antibodies.

Immunized Mouse Serum Titering

Enzyme-Linked Immunosorbent Assay (ELISA)

Antibody titering antigens were diluted with 1× phosphate-bufferedsaline (PBS) to 20 ng/μl and applied to 96-well with 50 μl (1 μg) eachwell, and antigen coated in 4° C. for overnight (12-16 hours). On thefollowing day that each well was washed 2 times with PBST (1×PBS, 0.1%Tween-20) 200 μl each, and blocked with 100 μl 1% Bovine serum albumin(BSA) for 2 hours in room temperature (about 25° C.). Then the wellswere washed by PBST 2 times with 200 μl each, and then the serialdilutions by 10 folds of test sera/antibodies were added, incubated in37° C. for 2 hours. After 2 hours incubation of antisera/antibodies withantigens, the 96-well plate was then washed by 200 μl PBST each well for4 times, and ensured to completely discard the solution from wells, thensecondary antibodies conjugated with horseradish phosphatase (HRP) wereadded to detect the bound primary antibodies. The secondary antibody wasused depend on the species of primary antibody; usually were anti- mouseIgG, or anti-rabbit IgG secondary antibodies. Secondary antibodies were1:5000 diluted in PB ST and 50 μl for each well, and incubated in 37° C.for 30 minutes. After incubation, the 96-well plate was washed by PBSTfor 4 times, and added with TMB, substrate for HRP, 50 μl for each andwait for visible color reaction and stop the reaction by another 50 μl2N H₂SO₄, then detect the OD values under 450 nm to calculate and plotthe curve for antibody specificity.

Selection of Antigen Specific Antibody Producing Hybridoma

Hybridoma culture media were acquired from Protech Inc., Taiwan, after1.5 - 2 months, and by ELISA to check antigen specific titers and selectfor positive clones. The 96-well plate was coated with antigens, pre-S₁region protein that were used for immunization, WT LHBS (pre-S₁+pre-S₂region) recombinant protein, pre-S₂ mutant (pre-S₁+pre-S₂ deletionmutant region) recombinant protein, and His-tagged E. coli recombinanthuman macrophage migration inhibitory factor (hMIF), used to rule outhis tag specificity. Antigen coated was 1 μg for each well and proteinswere diluted in PBS for 20 ng/μl and 50 μl for each well. Then thesehybridoma culture media were added as primary antibody, 100 μl for eachwell, and incubated under 37° C. for 2 hours. The following steps arethe same as above ELISA protocol for mouse antiserum titering.

Validation and Amplification of the Monoclonal Antibodies for the LHBSand the WT LHBS

The monoclonal antibodies for the LHBS and the WT LHBS were furtheramplified and characterized for their properties. Their sensitivitiesand specificities in the following experiments were evaluated by ELISA.In addition, to use these antibodies to develop a high-sensitiveimmunoassay kit, the purified LHBS and WT LHBS antibodies were alsodirectly conjugated with hydrogen peroxidase (HRP) for colorimetrydetection. The brief protocol for antibody conjugation to HRP is: Mixing2 mg of peroxidase and 100 uL of freshly prepared 0.1 M sodium periodateand stir the solution for 20 min at room temperature in the dark.Dialyze the modified enzyme against 1 mM sodium acetate buffer (pH 4.4)overnight at 4° C. Dissolve 4 mg of IgG in 500 uL of 10 mM sodiumcarbonate buffer (pH 9.5), and then adjust the pH of the dialyzed enzymesolution to 9.5 by using sodium borohydride solution. Stir the mixtureby gel filtration on a column of Sepharose CL-6B in PBS. Determine theA₂₈₀ and A₄₀₃. Pool the fractions in the first peak (both A₂₈₀ and A₄₀₃peaks coincide). Finally, add BSA to give a final concentration of 5mg/mL, and store the conjugate in aliquots at −20° C.

A lysis solution was prepared and used to release the HBV surfaceantigens from HBV viral particle in the serum. The lysis solution werecomposed of 2% HP-beta-CD (hydroxypropyl-beta-cyclodextrin), 0.5% TritonX-114, 0.5% Tween-20, 2 mM DTT (Dithiothreitol), and 1%2-Mercaptoethanol, all dissolved in 20 mM HEPES, pH 7. The sandwichELISA was processed with anti-HBS polyclonal antibodies coated onto96-well plate by 2 μg/ml and 50 μl each well as capture antibody. After4° C. overnight antibody coating step, the 96-well plate was then washedwith PBST (0.1% Tween-20 in phosphate buffered saline) 200 μl each wellfor 2 times and blocked with 1% BSA for 2-3 hours in room temperature(˜25° C.). With 1% BSA (Bovine serum albumin), the antibodies withnon-specific activities can be blocked by serum proteins. The blockingstep was then stopped and washed with PBST for 2-3 times for extra serumproteins to be removed.

Serum samples were 100-fold diluted in phosphate-buffered saline (PBS),and added into the 96-well with anti-HBS antibodies coated for 50 μleach. Then LHBS-specific and WT LHBS-specific monoclonal antibodiesworked as detection antibody were 1:5000 diluted in previous mentionedlysis solution and added into each well with diluted serum by 50 μl. TheLHBS and WT LHBS detection reaction was then incubated for 2 hours in37° C. The 96-well plate was later washed with 200 μl PBST (0.1%Tween-20 in PBS) each well for 5 times and 1:5000 PBS dilutedHRP-conjugated anti-mouse IgG antibodies were then added by 50 μl intoeach reaction well as secondary antibody and incubate in 37° C. foranother 1 hour. Finally, the reaction plate was washed with 200 μl PBSTeach well for 5 times and 50 μl HRP (Horseradish peroxidase) substrate,TMB, was added for visible color reaction with 37° C. incubation for5-10 minutes. And the OD values were detected by 450 nm on ELISA reader.

Development of an Immunoassay Kit to Detect the WT and Pre-S₂ DeletionMutant LHBS in Serum

Following purification and conjugation of the LHBS and the WTLHBS-specific monoclonal antibodies, these antibodies were used todevelop an immunoassay kit. Sera of 50 HBV carriers were recruited andused as positive control. Concentrations of the LHBS and the WT LHBS insera were determined by using the recombinant E. coli pre-S region WTand pre-S₂ deletion mutant proteins as the standards. The schematicrepresentation of the ELISA system is shown in FIG. 7. The levels of HRPconjugated to the LHBS- and WT LHBS-specific antibodies represent thoseof the LHBS and the WT LHBS, respectively. Therefore, the subtractedvalue of the LHBS by the WT LHBS in the same serum specimen was thelevel of the pre-S₂ deletion mutant in it.

Results

In the embodiment, the protein comprising the amino acids shown in SEQID NO: 1 is expressed in E. coli as recombinant protein, purified tohomogeneity then injected into mice for antibody generation. Afterseveral times of antibody boosting using the pre-S₁ region protein ofSEQ ID NO: 1, the mouse spleens were removed. The homogenizedsplenocytes were fused with myeloma cells to generate hybridomas, whichsecrete the anti-LHBS monoclonal antibodies. Hybridoma cells thatexpress high antibody titers are ip injected into mice for generation ofascites, where large amounts of antibodies exist. The monoclonalantibodies are purified from ascites, conjugated with horse radishperoxidase. The purified antibodies were employed to bind to the E. colirecombinant LHBS pre-S₁ region protein by ELISA assays. Monoclonalantibodies for the LHBS, the WT LHBS and the total HBS (including LHBS,MHBS and major HBS) have been developed. The results in FIG. 4 showedthat the LHBS antibody recognizes both the WT and pre-S₂ deletion mutantLHBS equally well with the sensitivity of 10 ng protein (FIG. 4, part A)and the antibody remains binding affinity to the LHBS up to 5×10⁴dilution (FIG. 4, part B). This antibody was large-scale amplified,purified and conjugated with HRP for further immunoassay use.

As for the WT LHBS-specific antibodies, the development processes issimilar to that of LHBS-specific antibody except the antigen being thepolypeptide of SEQ ID NO: 2, which will not further described here. Theresults in FIG. 5 showed that the WT LHBS antibody specificallyrecognizes the WT LHBS but not the pre-S₂ deletion mutant LHBS. Itsbinding sensitivity to the WT LHBS was approximate 30 ng (FIG. 5, partA) and the antibody remains binding affinity to the LHBS up to 10⁴dilution (FIG. 5, part B). This antibody has been large-scale amplified,purified and conjugated with HRP for ELISA use.

The monoclonal antibodies generated in the embodiments were employed toconstruct a sandwich ELISA system to detect the LHBS and WT LHBSproteins (FIG. 3). Using this assay kit, the binding specificity of theWT LHBS-specific antibody to the WT LHBS protein was detected in theWT/pre-S₂ deletion mutant LHBS mix. The results in FIG. 6 show that thepresence of the pre-S₂ deletion mutant does not appear to interfere thebinding of the WT LHBS-specific antibody to the WT LHBS protein,indicating that this ELISA system can detect the two LHBS types withhigh specificity.

The sandwich ELISA system developed in this study was used to detect theLHBS levels in sera of HBV carriers. Fifty HBsAg⁽⁺⁾ and 5 HBsAg⁽⁻⁾ sera,previously detected using the Abbott HBsAg detection system, wereinvestigated. The results found that all the HBsAg⁽⁺⁾ cases showedsignificantly higher levels of LHBS than the HBsAg⁽⁻⁾ cases (FIG. 7,part A). Analysis of the receiver operating characteristic (ROC) curveshowed that setting up the cut-off value of 0.02 ng LHBS/μl serum coulddistinguish the positive and negative cases with high sensitivity andspecificity (FIG. 7, part B). As for the detection of the pre-S₂deletion mutant LHBS, the signals detected in the LHBS and the WT LHBSas the respective amounts of proteins, are used to calculate the amountsof the pre-S₂ deletion mutant LHBS, which is detectable by theLHBS-specific but not the WT LHBS-specific antibodies (Table 1). Throughthis calculation, the amount of pre-S₂ deletion mutant LHBS wasobtained.

TABLE 1 Representative results of the ELISA detection for the wild typeand pre-S₂ mutant LHBS in human sera of HBV carriers. LHBS Wild typeLHBS Protein Protein Pre-S2 Average amount Average amount mutant Serumsample OD*1 OD*2 OD* (ng) OD*1 OD*2 OD* (ng) amount (ng) 1 0.21 0.230.22 0.39 0.27 0.32 0.29 0.40 0 2 1.17 1.14 1.16 2.70 0.45 0.52 0.490.99 1.71 3 0.32 0.32 0.32 0.63 0.09 0.07 0.08 0.01 0.62 4 0.71 0.700.70 1.59 0.14 0.14 0.14 0.13 1.46 5 0.37 0.37 0.37 0.75 0.11 0.11 0.110.06 0.69 6 0.25 0.25 0.25 0.45 0.18 0.19 0.19 0.24 0.21 Non-carrier0.07 0.06 0.06 0 0.05 0.04 0.04 0 0 *OD: optical density of lightabsorbance, which is generated by the ELISA reaction products.

Discussion

Hepatocellular carcinoma caused by chronic hepatitis B virus (HBV)infection is the most frequent visceral neoplasm worldwide. The virusinfection induces HCC through multiple pro-carcinogenic processes whichlead to cumulative genetic alterations in the long term period of virusbearing in carriers. In the HBV acute or immune-clearance infectionphase, liver manifests immune response-induced necroinflammation andconsequently hepatocyte regeneration. In the chronic hepatitis phase,the virus titers usually decrease, which attenuates liver inflammation,but it often accompanies with viral genome integration (10). Withcontinuous expression of viral genes from host genomes, a large portionof these chronic viral hepatitis progresses into liver cirrhosis anddysplasia and, ultimately, hepatocellular carcinoma. In this process,the viral proteins are believed to be important players, whichcross-talk with various host proteins to regulate the mechanism ofhepatocellular carcinogenesis (11). A number of HBV gene products havebeen identified as viral oncoproteins. The viral transactivating proteinHBx causes deregulation of cell cycle genes p53 and p21^(Waf1) andactivates the c-myc and c-jun oncoproteins (11,12). It also binds theDNA repair DNA damage-binding 1 (DDB1) protein and inhibits its activity(13). In addition, the pre-S₂ mutant LHBS, partially deleted ofapproximate 18 amino acids in the pre-S₂ region of the protein, wasfirst identified in the type II ground glass hepatocytes (GGH), whichwere the histological hallmarks of hepatocytes in advanced stages ofchronic HBV infection (2,3). It was (14, 15) found that HBV carriers whopresented with pre-S₂ deletion mutant LHBS in sera were predisposed toadvanced liver diseases, including liver cirrhosis and HCC. Our recentfindings (16) also indicated that type II GGH was correlated with theincreased tumor recurrence in and the decreased survival rates ofpatients with HCC undergoing hepatectomy. These findings invariablydemonstrated that pre-S₂ deletion mutant LHBS is a high-risk marker forHCC in chronic HBV carriers, as well as one for a poor prognosis inpatients with HCC.

In this study, we developed a sandwich ELISA system to detect the pre-S₂deletion mutant LHBS in sera. We previously developed a Pre-S Gene Chip,which detected the pre-S deletion based on DNA hybridization of the HBSgenes in patients to the DNA probes on the chip (9). However, due tothat the wild-type and pre-S mutant LHBS often co-exist in oneindividual HBV carrier, it often required the process of isolatingindividual FIBS clones to visualize the pre-S mutant ones. The ELISAsystem developed in this study has allowed us to directly detect thehigh-risk pre-S₂ deletion mutants in serum using a one-step detectionmethod, which has greatly improved the effectiveness. With thisconvenient tool, we will continue to detect the pre-S₂ mutant LHBS inlarger populations of HBV chronic carriers and to evaluate theassociation of pre-S₂ deletion mutant with clinical outcomes in variousstages of liver diseases including cirrhosis and HCC. A recent study hasalso found that the pre-S₂ deletion mutants increased after anti-viraltherapies in chronic HBV carriers, suggesting that the carrierspresenting the pre-S₂ mutant large HBV surface (LHBS) protein are theones in high risk to HCC (17). Therefore, the detection of pre-S₂deletion mutants in serum is important to predict HCC development orrecurrence after surgery, especially in the cases after anti-viraltreatments. The ELISA system developed in this study can provide a rapidand accurate method for detecting deletions in the HBV Pre-S region,thereby assessing a HBV carrier's risk of developing cirrhosis/HCC

In an embodiment, patients who are HBeAg-negative and with viral loadhigher than 10⁴ copies/ml are considered to be high risk groups. Thesepatients should be tested for pre-S₂ deletion mutants. Briefly, LHBSprotein and WT LHBS protein in patients who are HBeAg-negative withviral load higher than 10⁴ copies/ml were detected by the ELISA methoddescribed above, and the amount of the pre-S₂ deletion mutant LHBSprotein was calculated by subtracting the amount of the WT LHBS proteinfrom that of the LHBS protein, as shown in Table 1. If the patients areHBV carriers with the pre-S₂ deletion mutant LHBS protein, the patientswould be considered as high risk group of developing hepatocellularcarcinoma (HCC) or cirrhosis, comparing to those who don't.

In another embodiment, patients who have persistence of surface antigensor HBV DNA after antiviral therapy for 3 months represent the presenceof pre-S₂ deletion mutants and the high risk group to develop HCC. Thesepatients should be tested for serum HBV pre-S₂ deletion mutants.Similarly, LHBS protein and WT LHBS protein in patients who havepersistence of surface antigens or HBV DNA after antiviral therapy for1-12 month(s), preferably 1-5 month(s), more preferably 3 months, weredetected by the ELISA method described above, and the amount of thepre-S₂ deletion mutant LHBS protein was calculated by subtracting theamount of the WT LHBS protein from that of the LHBS protein, as shown inTable 1. If the patients after antiviral therapy carry the pre-S₂deletion mutant LHBS protein, the patients would be considered as highrisk group of developing hepatocellular carcinoma (HCC) or cirrhosis,comparing to those who don't.

In still another embodiment, patients receiving hepatectomy surgery forHBV-related HCC will be screened for the pre-S₂ deletion mutants inserum 3 months after surgery. Evaluating ground glass hepatocytes willbe done simultaneously to assess the risk of HCC recurrence or de novoHCC development. The presence of pre-S₂ deletion mutants in serum andGGHs in liver tissues are considered to have increased risk of HCCrecurrence. Similarly, LHBS protein and WT LHBS protein in patientsreceiving hepatectomy surgery for HBV-related HCC were detected by theELISA method described above, and the amount of the pre-S₂ deletionmutant LHBS protein was calculated by subtracting the amount of the WTLHBS protein from that of the LHBS protein, as shown in Table 1. If thepatients after surgery carry the pre-S₂ deletion mutant LHBS protein,the patients would be considered as high risk group of cancerrecurrence, comparing to those who don't.

While the means of specific embodiments in present invention has beendescribed by reference drawings, numerous modifications and variationscould be made thereto by those skilled in the art without departing fromthe scope and spirit of the invention set forth in the claims. Themodifications and variations should in a range limited by thespecification of the present invention.

What is claimed is:
 1. A method of detecting a pre-S₂ deletion mutantlarge hepatitis B virus surface protein in a biological sample,comprising: incubating a biological sample with a first antibodyimmobilized on a substrate to bind a hepatitis B virus surface proteinto the first antibody, wherein the first antibody specifically binds toamino acid residues 1-226 of hepatitis B virus surface protein;detecting a large hepatitis B virus surface protein and a wild typelarge hepatitis B virus surface protein bound to the immobilized firstantibody by incubating the bound hepatitis B virus surface protein witha second antibody specifically binding to amino acid residues 1-119 oflarge hepatitis B virus surface protein and a third antibodyspecifically binding to amino acid residues 125-142 of large hepatitis Bvirus surface protein, respectively; and calculating the amount of thepre-S₂ deletion mutant large hepatitis B virus surface protein bysubtracting the amount of the wild type large hepatitis B virus surfaceprotein from that of the large hepatitis B virus surface protein.
 2. Themethod of claim 1, wherein the first antibody is a hepatitis B virussurface protein-specific antibody produced by a hybridoma cell linedeposited on Oct. 22, 2014 under accession number BCRC960491 at the FoodIndustry Research and Development Institute, 331 Shih-Pin Road, Hsinchu300, Taiwan, and also on Oct. 31, 2014 under accession number DSMACC3252 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig,Germany, or progeny thereof, the second antibody is a large hepatitis Bvirus surface protein-specific antibody produced by a hybridoma cellline deposited on Sep. 1, 2014 under accession number BCRC960490 at theFood Industry Research and Development Institute, 331 Shih-Pin Road,Hsinchu 300, Taiwan, and also on Oct. 31, 2014 under accession numberDSM ACC3253 at Leibniz-Institut DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124Braunschweig, Germany, or progeny thereof, and the third antibody is awild type large hepatitis B virus surface protein-specific antibodyproduced by a hybridoma cell line deposited on Jul. 16, 2014 underaccession number BCRC960488 at the Food Industry Research andDevelopment Institute, 331 Shih-Pin Road, Hsinchu 300, Taiwan, and alsoon Oct. 31, 2014 under accession number DSM ACC3251 at Leibniz-InstitutDSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ),Inhoffenstr. 7B, 38124 Braunschweig, Germany, or progeny thereof.
 3. Themethod of claim 1, wherein the biological sample is isolated from ahuman.
 4. The method of claim 3, wherein the human is a HBV carrier or aliver cancer patient.
 5. The method of claim 4, wherein the HBV carrieris HBeAg-negative, with HBV viral load higher than 10⁴ copies/ml of thebiological sample, or the combination thereof.
 6. The method of claim 5,further comprising a step of assessing a risk of developinghepatocellular carcinoma or cirrhosis of the HBV carrier.
 7. The methodof claim 4, wherein the HBV carrier has undergone an antiviral therapyusing nucleoside analogs or interferon drugs.
 8. The method of claim 7,further comprising a step of assessing a risk of developinghepatocellular carcinoma or cirrhosis after the antiviral therapy. 9.The method of claim 4, wherein the liver cancer patient is ahepatocellular carcinoma patient undergone a hepatectomy surgery. 10.The method of claim 9, further comprising a step of assessing risk ofcancer recurrence of the hepatocellular carcinoma patient at 1 to 12month(s) after the surgery.
 11. The method of claim 3, wherein thebiological sample is whole blood, plasma or serum.
 12. The method ofclaim 1, further comprising a step of isolating hepatitis B virussurface protein from the biological sample.